Herpes simplex virus (HSV) causes lifelong latent infections in humans. It is responsible for significant disease, ranging from cold sores and genital infections to blindness and fatal encephalitis. The long- term goal of this project is to understand the molecular mechanisms that HSV uses to gain entry into host cells. An emerging concept in herpesvirology is that endosomal pH of the host cell is required for viral entry, often in a cell type specific manner. However, the mechanistic role that low pH plays in herpes viral entry is not clear. HSV utilizes a low pH, endocytic pathway for infection of epithelial cells, the primary portal of entry into the human host and the site of recurrent infection. HSV gB belongs to the class III viral fusion protein family, whose members also drive the entry several other important enveloped viruses. The class III fusion mechanism remains poorly defined. The focus of this proposal is the delineation of the mechanism of low pH membrane fusion mediated by HSV. Based on previous results and our new preliminary studies, we have formulated three specific aims. In Specific Aim # 1, we will elucidate viral and cellular parameters of HSV membrane fusion triggered by low pH. We will reveal critical regions of the fusion protein gB and define the roles of cellular receptors in cell-cell fusion triggered by low pH. In Aim 2, we will delineate a novel role for an HSV envelope protein, not previously associated with fusion and entry. We will elucidate its importance for conformational changes triggered by acidic pH. For Aim 3, we will determine the functional consequences of the interaction between HSV gH/gL and gB in the context of low pH fusion and entry. Our experimental design employs techniques of molecular virology, biochemistry and cell biology. Achieving these aims will fill critical knowledge gaps about how the complex fusion mechanism of herpesviruses is triggered by host intravesicular pH in physiologically relevant cell types. The results will represent significant advances in our understanding of class III fusion mechanisms, and may aid in development of novel, antiviral interventions.